Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea worldwide and is associated with high rates of morbidity and mortality. The mechanisms underlying EPEC pathogenesis are not understood. EPEC has direct effects on host intestinal epithelial functions including tight junction (TJ) permeability which is believed to contribute to diarrhea. The objective of this proposal is to elucidate the cellular and molecular basis for the EPEC-induced alterations in host intestinal epithelial tight junction barrier function. In part, the changes in intestinal TJ permeability are related to contraction of the perijunctional cytoskeletal ring. EPEC also alters TJ-associated proteins, including occludin, ZO-1, and claudin-1. Several TJ proteins directly interact with the cytoskeleton thus reconciling the observation that EPEC may exploit both mechanisms by which the TJ barrier is regulated. Experiments for Specific Aim 1 will characterize the effects of EPEC on individual TJ proteins and their interactions with each other. The focus of Specific Aim 2 will be to investigate the relationship between the two mechanisms by which EPEC perturbs the TJ barrier i.e., cytoskeletal contraction and alterations in TJ proteins. The proteins that transmit signals initiated by EPEC attachment to the host cell are not defined. Ezrin, a membrane-cytoskeleton linker molecule capable of mediating signal transduction events, may be involved in the cross-talk between microbe and host. Specific Aim 3 will define the role of the membrane-cytoskeleton linker protein ezrin in EPEC-induced alterations in tight junctions. EPEC virulence genes are likely involved in the perturbation of TJ barrier function since non- pathogenic E. coli do not elicit the same effect. The pathogenicity island of EPEC, called the locus of enterocyte effacement, has been sequenced and cloned and contains genes encoding type III secretory machinery, through which bacteria directly deliver proteins into host cells. Studies outlined in Specific Aim 4 will determine the specific EPEC proteins involved in the alteration of host intestinal epithelial TJ proteins and identify the signaling pathways responsible. These Specific Aims will address the overall hypothesis of this proposal which is that EPEC disrupts the tight junction barrier by stimulating contraction of the perijunctional cytoskeletal ring and by altering tight junction-associated proteins via signaling pathways transduced by the membrane-cytoskeletal linker protein ezrin. We further hypothesize that specific EPEC attachment factors and/or injection of proteins into host intestinal epithelial cells by type III secretion are responsible for this physiological alteration.